F13A1 Expression Research Articles

MicroRNA-1271-5p suppresses the proliferation, invasion and migration of oral squamous cell carcinoma by inhibiting F13A1 expression

MicroRNA-1271-5p suppresses the proliferation, invasion and migration of oral squamous cell carcinoma by inhibiting F13A1 expression

Transglutaminases are oncogenic biomarkers in human cancers and therapeutic targeting of TGM2 blocks chemoresistance and macrophage infiltration in pancreatic cancer.

Transglutaminases (TGs) are multifunctional enzymes exhibiting transglutaminase crosslinking, as well asatypical GTPase/ATPase and kinase activities. Here, we usedan integrated comprehensive analysis to assess the genomic, transcriptomic and immunological landscapes of TGs across cancers. Gene expression and immune cell infiltrationpatterns across cancers were obtained from The Cancer Genome Atlas (TCGA) database and Gene Set Enrichment Analysis (GSEA) datasets. Western blotting, immunofluorescence staining, enzyme-linked immunosorbent assays, and orthotopic xenograft models were used to validate our database-derived results. We found that theoverall expression of TGs (designated as the TG score) is significantly upregulated in multiple cancers and related to aworse patient survival. The expression of TG family members can be regulated through multiple mechanisms at the genetic, epigenetic and transcriptional levels. The expression of transcription factors crucial for epithelial-to-mesenchymal transition (EMT) is commonly correlated with the TG score in many cancer types. Importantly, TGM2 expression displays a close connection with chemoresistance to a wide range of chemotherapeutic drugs. We found thatTGM2 expression, F13A1 expression and the overall TG score were positively correlated with the infiltration of immune cells in all cancer types tested. Functional and clinical verification revealed that ahigher TGM2 expression is linked with aworse patient survival, an increased IC50 value of gemcitabine, and a higher abundance of tumor-infiltrating macrophages in pancreatic cancer. Mechanistically, we found thatincreased C-C motif chemokine ligand 2 (CCL2) release mediated by TGM2 contributes to macrophage infiltration into the tumor microenvironment. Our results reveal the relevance and molecular networks of TG genes in human cancers and highlight the importance of TGM2 in pancreatic cancer, which may provide promising directions for immunotherapy and foraddressing chemoresistance.

ACQUIRED FACTOR XIII DEFICIENCY WITH RUNX1 MUTATION, A REPORT OF TWO CASES TREATED WITH FACTOR XIII CONCENTRATE

Acquired FXIII deficiency has been described in association with malignancies or autoimmune disorders. We report two cases of acquired FXIII deficiency associated with hematologic malignancies. The first patient is a 60-year-old male with CMML who presented 4 weeks after confirming his diagnosis with non-traumatic anterior abdominal wall hematoma. Workup revealed FXIII deficiency. He was treated with FXIII replacement and other supportive measures. The hematoma resolved and patient was maintained on factor replacement. Unfortunately, his disease transformed to AML and he succumbed to death after starting AML therapy despite achieving complete remission. The second patient is a 24-year-old male patient post haploidentical transplant for intermediate risk AML. He developed hemorrhagic cystitis day 36 post-transplant and non-traumatic subdural hematoma on day 60 post-transplant. Workup revealed FXIII deficiency. He was treated with factor replacement and the subdural hematoma resolved with improvement of the hemorrhagic cystitis. Both patients had RUNX1 mutation which regulates expression of F13A1 in megakaryocyte this can decreased platelet expression of F13A1 in patient with RUNX1 haplodeficiency which lead to platelet dysfunction. FXIII deficiency should be considered for patient with unexplained bleeding with normal routine workup.

F13A1 transglutaminase expression in human adipose tissue increases in acquired excess weight and associates with inflammatory status of adipocytes.

F13A1/FXIII-A transglutaminase has been linked to adipogenesis in cells and to obesity in humans and mice, however, its role and associated molecular pathways in human acquired excess weight have not been explored. We examined F13A1 expression and association to human weight gain in weight-discordant monozygotic twins (Heavy-Lean difference (ΔWeight, 16.8 kg ± 7.16 for n = 12). The twin pairs were examined for body composition (by dual-energy X-ray absorptiometry), abdominal body fat distribution (by magnetic resonance imaging), liver fat content (by magnetic resonance spectroscopy), circulating adipocytokines, leptin and adiponectin, as well as serum lipids. Affymetrix full transcriptome mRNA analysis was performed from adipose tissue and adipocyte-enriched fractions from subcutaneous abdominal adipose tissue biopsies. F13A1 differential expression between the heavy and lean co-twins was examined and its correlation transcriptome changes between co-twins were performed. F13A1 mRNA showed significant increase in adipose tissue (p < 0.0001) and an adipocyte-enriched fraction (p = 0.0012) of the heavier co-twin. F13A1 differential expression in adipose tissue (Heavy-Lean ΔF13A1) showed significant negative correlation with circulating adiponectin (p = 0.0195) and a positive correlation with ΔWeight (p = 0.034), ΔBodyFat (0.044) and ΔAdipocyte size (volume, p = 0.012;) in adipocyte-enriched fraction. A whole transcriptome-wide association study (TWAS) on ΔF13A1 vs weight-correlated ΔTranscriptome identified 182 F13A1-associated genes (r > 0.7, p = 0.05) with functions in several biological pathways including cell stress, inflammatory response, activation of cells/leukocytes, angiogenesis and extracellular matrix remodeling. F13A1 did not associate with liver fat accumulation. F13A1 levels in adipose tissue increase with acquired excess weight and associate with pro-inflammatory, cell stress and tissue remodeling pathways. This supports its role in expansion and inflammation of adipose tissue in obesity.

Transcription Factor RUNX1 Regulates Factor FXIIIA Subunit (F13A1) Expression in Megakaryocytic Cells and Platelet F13A1 Expression is Downregulated in RUNX1 Haplodeficiency

Plasma Factor XIII, a transglutaminase, is a complex of two A (gene F13A1) chains with catalytic activity and two B chains (gene F13B). FXIII induces clot stabilization and retraction via cross-linking of fibrin monomers and other proteins. Factor XIII deficiency in plasma causes a major bleeding disorder with impaired clot retraction. FXIIIA is synthesized in megakaryocytes, and is abundant in platelets. It is expressed on activated platelets and cross-links proteins on platelet surface. Little is known regarding the regulation of F13A1 in megakaryocytes and platelets. RUNX1 is a major hematopoietic transcription factor and regulates expression of numerous genes in megakaryocytes and platelets. Patients with RUNX1 haplodeficiency (RHD) with heterozygous mutations have thrombocytopenia, platelet dysfunction with impaired responses to activation, and predisposition to myeloid malignancies. Platelet expression profiling using Affymetrix microarrays of a patient (P1) with a heterozygous RUNX1 mutation (c.969-323G&amp;gt;T) revealed decreased F13A expression, which encodes for subunit A of FXIII: fold-change 0.30; p=0.006; patient profiled twice 10 months apart and compared to 6 healthy subjects. These findings were confirmed by qPCR With RT-PCR platelet F13A mRNA expression was decreased to ~ 20% of mean expression in 4 subjects. Platelet F13A protein expression was decreased by ~50% as assessed by corrected total cellular fluorescence imaging. Platelet F13A mRNA expression was also decreased (to less than 20% of expression in 5 controls) in two siblings (P2 and P3 ages 8 and 3 years) from an unrelated family with a heterozygous RUNX1 mutation 508+1G&amp;gt;A). Plasma FXIII was normal in the patient P1. We studied the regulation of F13A by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic transformation. In silico analyses of the 5' upstream region of F13A showed 7 RUNX1 consensus sites within 545bp from ATG. Chromatin immunoprecipitation studies performed using HEL cells and RUNX1 antibody showed that that RUNX1 binds in the region encompassing site 4 and sites 5-7. In parallel, electrophoretic mobility shift assay (EMSA) studies showed binding of nuclear protein to labeled DNA probes with sites 1, 4, 5, 6, and 7. RUNX1 antibody produced a "supershift" with addition suggesting RUNX1 binding. Site-directed mutagenesis of the RUNX1 binding sites in F13A1 promoter decreased promoter activity in luciferase reporter studies in HEL cells, providing evidence that the binding sites are functional. Further, overexpression of RUNX1 in HEL cells increased F13A1 promoter activity and protein while siRNA RUNX1 knockdown reduced F13A1 protein. We assessed clot retraction over 90 min at 37oC in the patient P1 and his daughter (also with RUNX1 mutation) in citrated whole blood following addition of tissue factor (1 pM) and CaCl2 (1 mM), and in washed platelets resuspended in platelet poor plasma from a healthy subject supplemented with added fibrinogen (0.5 mg/mL) and stimulated with 0.5 U/mL thrombin in the presence of CaCl2. These preliminary studies suggested that clot retraction was slower in the patient P1 compared to the controls, particularly in studies with platelet suspensions. Conclusions: Overall, these studies provide the first evidence that hematopoietic transcription factor RUNX1 regulates expression of F13A1 in megakaryocytic cells and that platelet expression of F13A1 is decreased in platelets from patients with RUNX1 haplodeficiency. The decreased expression of F13A1 in RHD may contribute to the platelet dysfunction in RHD. These findings are particularly interesting because they reflect regulation of a coagulation protein (FXIIIA) by hematopoietic transcription factor RUNX1. Disclosures Lambert: Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy; Octapharma: Consultancy, Research Funding; Bayer: Consultancy; Platelet Disorder Support Association (PDSA): Consultancy; 22qSociety: Consultancy; ITP Australia: Consultancy; CdLS Foundation: Consultancy; RDMD ITP study: Consultancy; Sysmex: Research Funding; AstraZeneca: Research Funding; Argenix: Consultancy; ClinGen: Honoraria.

Transglutaminases and Obesity in Humans: Association of F13A1 to Adipocyte Hypertrophy and Adipose Tissue Immune Response.

Transglutaminases TG2 and FXIII-A have recently been linked to adipose tissue biology and obesity, however, human studies for TG family members in adipocytes have not been conducted. In this study, we investigated the association of TGM family members to acquired weight gain in a rare set of monozygotic (MZ) twins discordant for body weight, i.e., heavy–lean twin pairs. We report that F13A1 is the only TGM family member showing significantly altered, higher expression in adipose tissue of the heavier twin. Our previous work linked adipocyte F13A1 to increased weight, body fat mass, adipocyte size, and pro-inflammatory pathways. Here, we explored further the link of F13A1 to adipocyte size in the MZ twins via a previously conducted TWA study that was further mined for genes that specifically associate to hypertrophic adipocytes. We report that differential expression of F13A1 (ΔHeavy–Lean) associated with 47 genes which were linked via gene enrichment analysis to immune response, leucocyte and neutrophil activation, as well as cytokine response and signaling. Our work brings further support to the role of F13A1 in the human adipose tissue pathology, suggesting a role in the cascade that links hypertrophic adipocytes with inflammation.

2029-P: Role of Adipose Tissue Macrophage–Derived F13A1 in Regulating Preadipocyte Hyperplasia during Obesity

The epidemic of DM has increased in the last decades and is the leading cause of decreased life expectancy. AT adapts to nutrient excess by balancing hypertrophy and hyperplasia that contribute to AT expansion. Unhealthy AT expansion seen in a person with diabetes is characterized by decreased preadipocyte (PA) content, and increased adipocyte size. AT macrophages (ATM) contribute to unhealthy AT expansion by blocking adipogenesis, promoting adipocyte hypertrophy, fibrosis and insulin resistance (IR). Many of the cellular and molecular mechanisms by which adipocyte numbers and size is regulated by ATMs in vivo are still unknown, especially in human obesity. This study focused on the hypotheses that ATM expression of F13A1, a tissue transglutaminase enzyme shown to regulate fibrin networks during tissue growth: 1) modulates abundance of preadipocyte (PA) populations during obesity and DM states, and 2) contributes to adipocyte hypertrophic states and potential metabolic dysfunction. The premise for these studies was based on evidence that F13A1 is increased in Visceral AT (VAT) of DM individuals compared to non-DM and that it is produced by a tissue remodeling ATM subset increased in DM. We utilized nondiabetic (NDM) and DM human secondary PA treated with F13A1 and assessed the following: 1) F13A1 expression requirement for the inhibition of healthy PA expansion in obesity, and 2) the mechanisms by which its function leads to arrested proliferation. Results showed that F13A1 impairs PA proliferation through its exerted effects on PA ECM fibronectin crosslink leading to: 1) downregulation of downstream gene proliferative signaling pathways and 2) defaulted adipocyte differentiation with hypertrophic profiles as an antagonistic response to decreased proliferation. These studies have advanced our understanding of ATM-derived F13A1 as a potential immunotherapeutic target to improve AT function. Disclosure D.M. Elizondo: None. L.M. Geletka: None. C.G. Flesher: None. C.N. Lumeng: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (T32DK101357)

Coagulation FXIII-a Protein Expression Defines Three Novel Sub-Populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized By Distinct Gene Expression Signatures

Background Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). FXIII-A expression determined by flow cytometry (FC) exhibited characteristically distinct expression patterns in subgroups of lymphoblasts. The FXIII-A negative subgroup was significantly associated with the 'B-other' genetic category and had an unfavorable disease outcome. Methods RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology (GO) analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed (DE) genes were validated by RT-Q-PCR. Results We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the expression of FXIII-A protein, as determined by FC. We observed three well-defined categories of FXIII-A protein expression: FXIII-A negative (&lt;20% FXIII-A positive blasts), FXIII-A dim (20-80% FXIII-A positive blasts), and FXIII-A bright (&gt;80% FXIII-A positive blasts). The intensity of the FXIII-A expression increased continuously, which excluded the existence of a distinct FXIII-A negative and a FXIII-A bright subpopulation within the FXIII-A dim subgroup (Image 1/A). These three subgroups defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. There were 26 DE genes found when comparing the FXIII-A negative with the FXIII-A bright subgroup. The FXIII-A dim vs. bright comparison resulted in 155 DE genes and there were 88 DE genes identified between the FXIII-A negative and dim subgroups (Image 1/B). Expression of F13A1 gene was detected and readily validated by RT-Q-PCR in every sample. Intensity of gene expression; however, was characteristically different among samples of the three different FXIII-A protein expression subgroups with an increasing intensity in terms of relative fold-changes measured by RT-Q-PCR from the FXIII-A negative, through dim to bright subgroups. We selected 13/45 genes based on fold-change results detected by microarray, whereas an additional 32/45 genes were selected according to enriched functional categories of potential interest as defined by the GO analysis. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2, validated by RT-Q-PCR according to the FXIII-A status, followed the pattern of intensity of the expression of the F13A1 gene. Of these genes, ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 appear to have a role in leukemia and other forms of cancer. NUP43 has not yet been shown to be associated with any forms of human cancer in contrast to other members of the NUP gene family. PLAC8 which is a trophoblast lineage marker was most intensively expressed in the FXIII-A dim subgroup. This gene has been shown to be aberrantly activated in various types of cancer arising in mammals and mammalian cancer cell lines, but not in any subtype of human ALL (Image 1/C). Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of 'B-other' samples. We identified 14 DE genes by microarray overlapping with DE genes of the 'B-other' subgroup defined by the COALL Group. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the 'B-other' subgroup. Conclusions We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL. Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Validated genes proved biologically and clinically relevant. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment. Figure Disclosures No relevant conflicts of interest to declare.

Coagulation FXIII-A Protein Expression Defines Three Novel Sub-populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized by Distinct Gene Expression Signatures.

Background: Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). Flow cytometry (FC) revealed three distinct expression patterns, i.e., FXIII-A negative, FXIII-A dim, and FXIII-A bright subgroups. The FXIII-A negative subgroup was significantly associated with the “B-other” genetic category and had an unfavorable disease outcome.Methods: RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28,869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed genes were validated by RT-Q-PCR.Results: We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the FXIII-A protein expression subgroups which defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 followed the pattern of change in the intensity of the expression of the F13A1 gene. Common enhancer elements of these genes revealed by in silico analysis suggest that common transcription factors may regulate the expression of these genes in a similar fashion. PLAC8 was downregulated in the FXIII-A bright subgroup. Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of “B-other” samples. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the “B-other” subgroup. Validated genes proved biologically and clinically relevant. We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL.Conclusions: Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment.

Comprehensive Metabolomics Analysis of Xueshuan Xinmaining Tablet in Blood Stasis Model Rats Using UPLC-Q/TOF-MS.

Blood stasis syndrome (BSS) is one of the most common Chinese medicine patterns in coronary heart disease. Our previous work proved that Xueshuan Xinmaining Tablet (XXT) could treat blood stasis through regulating the expression of F13a1, Car1 and Tbxa2r. In the current study, the effect and mechanism of XXT on BSS was comprehensively and holistically investigated based on a metabolomics approach. Urine and plasma samples of 10 BBS rats treated with XXT (XT), 9 BSS model rats (BM) and 11 normal control (NC) rats were collected and then determined by UPLC-Q/TOP-MS. Multivariate analyses were applied to distinguish differentiate urinary and plasma metabolite patterns between three groups. Results showed that a clear separation of three groups was achieved. XT group was located between BM group and NC group, and showing a tendency of recovering to NC group, which was consistent with the results of hemorheological studies. Some significantly changed metabolites like cortexolone, 3α,21-dihydroxy-5β-pregnane-11,20-dione and 19S-hete and leukotriene A4, chiefly involved in steroid hormone biosynthesis, arachidonic acid metabolism and lipid metabolism, were found and identified to explain the mechanism. These potential markers and their corresponding pathways will help explain the mechanism of BSS and XXT treatment. This work also proves that metabolomics is effective in traditional Chinese medicinal research.

Xueshuan Xinmaining Tablet Treats Blood Stasis through Regulating the Expression of F13a1, Car1, and Tbxa2r.

Xueshuan Xinmaining Tablet (XXT), the Chinese formula, has long been administered in clinical practice for the treatment of cerebral thrombosis and coronary heart disease. In this study, we aimed to study the effect and the molecular mechanism of activating blood circulation and removing blood stasis. Rat models of cold coagulation blood stasis were induced with ice-water bath and epinephrine to assess the amelioration of blood stasis by XXT. Microarray technique was used to identify gene expression from the model and XXT-treated rats. In addition, Quantitative Real-Time PCR (qPCR) was performed to verify the microarray results. The results showed that XXT had a good therapeutic effect on blood stasis by reducing the whole blood viscosity (WBV), plasma viscosity (PV), increasing PT, APTT and TT, and by inhibiting platelet aggregation. Genes were differentially expressed in rats among the model group and the XXT-pretreated groups. XXT ameliorated blood stasis by regulating the expressions of F13a1, Car1, and Tbxa2r.

Subunit a of Coagulation Factor XIII As a New Biomarker in Childhood Acute Lymphoblastic Leukemia?

Background: Using multiparameter flow cytometry (FC), Western blot, ELISA and laser scanning microscopy, leukemic B-cell progenitor (BCP) lymphoblasts were identified as a novel expression site of coagulation factor XIII subunit A (FXIIIA; Kiss F. et al. Thomb Hemost, 2006.).Objectives: The significance of FXIIIA expression, defined by FC, on the clinical outcome of children with BCP acute lymphoblastic leukemia (ALL) was studied. We used gene expression profile (GEP) analysis and pathway analysis to identify genes which may contribute to the phenotype of the F13a1 low expression patient subgroup. These genes will be tested for the presence of mutations by next generation sequencing in the next phase of the study.Method: ALL immunophenotyping, including FXIIIA expression and detection of minimal residual disease (MRD) in Day 15 bone marrow was determined by four color FC analysis. Clinical course of FXIIIA-positive and –negative children with ALL was retrospectively studied and 3-yr overall survival (OS) data were calculated and compared by Kaplan-Mayer plot analysis. We analyzed 3 public datasets in the GEO Database (GSE47051, GSE13351 and GSE13425), including the dataset of the University of Padova (GSE47051) containing data on GEPs of children with ALL using GeneSpring and Ingenuity Pathway Analysis softwares.Results: Sixty-three per cent of BCP ALL cases, investigated by FC at the Department of Laboratory Medicine, University of Debrecen expressed FXIIIA. Three-year overall survival (OS) of children with FXIIIA-positive ALL was significantly higher (87%) than 3-yr OS of patients with FXIIIA-negative ALL (65%). As expected, Day 15 FC-MRD successfully separated patients into three well-defined groups with different OS rates: 92%, 72% and 43% in FC-MRD SR, IR and HR groups, respectively. FXIIIA expression has merged FC-MRD SR and IR groups with a significant difference between FXIIIA-positive vs. -negative patients.In the GeneSpring analysis, patients were separated into two groups: F13a1 low expression, and F13a1 high expression group, with at least 2-fold difference in their F13a1 expression level. F13a1 low and high expression groups exhibited a characteristically different GEP (at least 2-fold difference, p≤0.05, t-test, Benjamini-Hochberg correction for FDR). Low F13a1 expression level was prevalent among the genetic subgroup of “B-other” samples, high F13a1 expression level was associated with the t(1;19) genetic subgroup of childhood ALL. We found 8 genes that were significantly down-regulated, and one, that was upregulated in the F13a1 low expression group in all three datasets (Table 1), and 28 similarly deregulated genes in at least two of the three datasets. We identified two chromosomal loci, 19p13.3 and 16q22, with 11 and 2 deregulated genes, respectively, within the F13a1 low expression group (Table 1). Using Ingenuity Pathway Analysis, we identified a network of genes participating in B cell development that were deregulated in the F13a1 low expression group. Using the Ingenuity upstream regulator analysis, deregulation of NUPR1, TCF3 and IKZF1 were predicted in the F13a1 low expression group.Conclusion: FXIIIA expression by FC may define a new subgroup of childhood ALL, with a partial overlap in the GEPs of the F13a1 low expression group and the recently defined BCR-ABL1-like group (Table 1). FXIIIA expression by FC may help in selecting those cases which require a more sophisticated – and more expensive - genetic diagnosis to define the optimal risk-tailored therapy. In addition, FXIIIA may become a useful marker for Day 15 MRD detection.Grant sponsor TÁMOP 4.2.2.A-11/1/KONV-2012-0025 - the project is co-financed by the European Union and the European Social Fund; The AIRC (Associazione Italiana Ricerca su Cancro) project; OTKA K108885. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression Level Of A Coagulation Factor Gene, F13A1 Defines Characteristic Subpopulations Of Childhood Acute Lymphoblastic Leukemia

Using multiparameter flow cytometry, Western blot, ELISA and laser scanning microscopy, leukemic B-cell progenitor lymphoblasts were identified as a novel expression site of coagulation factor XIII subunit A (FXIIIA) (Kiss et al Thromb Haemost 2006). Three-year overall survival (OS) of children with FXIIIA-positive acute lymphoblastic leukemia (ALL) was significantly higher (87%) than 3-yr OS of patients with FXIIIA-negative ALL (65%). Here we report on gene expression profiles (GEP) associated with FXIIIA-positive vs. FXIIIA–negative childhood (ch)ALL. Finnally, the expression levels of F13A1 gene in cytogenetic subgroups were investigated.GEP of 11 FXIIIA-positive and 9 FXIIIA-negative samples of patients treated at the Department of Hematology-Oncology, University of Debrecen was investigated using HG-U 133 plus 2.0 Affymetrix Platform Array. Expression sequence tags (EST) characterized by expression levels with at least 2 logs difference among FXIIIA-positive vs. FXIIIA-negative samples were selected and validated with real-time quantitative PCR. Extending the analysis, we have searched the database containing GEPs of 317 children with ALL treated at the Division of Hemato-Oncology, Department of Women's and Children's Health, University of Padova, using R package and Partek Genomic Suite softwares.FXIIIA-positive and FXIIIA-negative samples exhibited a characteristically different GEP. In the FXIIIA-positive samples one of the overexpressed genes was F13A1. FXIIIA-negative samples contained two characteristic groups of overexpressed genes. One of these consisted of genes participating in B-cell development: EBF1, IKZF1 and PAX5. The second set consisted of genes encoding for tyrosine kinases: JAK1, JAK3, NC07523, NC08002, NC06523, NC08345 and for serine-threonine kinase NC00027. In contrast, FXIIIA-positive samples contained only two overexpressed tyrosine kinases: C-KIT and JAK2.A wide variation of F13A1 expression levels of the 317 Padova patients was observed. Since expression level of FXIIIA protein of these patients has not been determined, we have arbitrarily defined F13A1 gene expression levels below 106 as “low” and gene expression levels exceeding 109 as “high”. Considering these two groups, we have investigated the distribution of F13A1 expression among the known cytogenetic subgroups of ALL. Low F13A1 expression was prevalent among “B-other” samples, high F13A1 expression was associated with t(1;19).The pattern of overexpressed ESTs, accumulation of low F13A1 expressing samples in the “B-other” cytogenetic group of ALL and the unfavorable disease outcome of FXIIIA-negative cases may suggest a possible overlap between FXIIIA-negative ALL and BCR-ABL1-like ALL identified recently in the “B-other” group. The nature and extent of this overlap will be investigated prospectively in the BFM ALL-IC 2009 clinical trial. In contrast, high expression levels of F13A1 accumulated preferentially within the t(1;19) genetic group, associated with a good prognosis. Detection of FXIIIA expression by flow cytometry may offer an easy and non-expensive tool for defining new prognostic subpopulations of ALL. Grant supportTÁMOP 4.2.2.A-11/1/KONV-2012-0025 project (CK, KG, HZ, IG, IS and JK). The project is co-financed by the European Union and the European Social Fund and the AIRC (Associazione Italiana Ricerca su Cancro; SB) project. Disclosures:No relevant conflicts of interest to declare.

Pro‐atherogenic effects of probucol may be mediated through alterations in the coagulation system in apo E‐KO mice

We have previously shown that probucol paradoxically increases atherogenesis in apo E‐KO mice, while dietary phytosterol significantly reduce the disease in this animal model. Male apo E‐KO mice were fed with an atherogenic diet containing 9% (w/w) fat and 0.2% (w/w) cholesterol in the presence of either 2% (w/w) dietary phytosterol mixture (phytosterol‐treated group, n=8), or 1% (w/w) probucol (probucol‐treated group, n=8) or nothing (control group, n=8) for 18 weeks. Plasma total cholesterol levels were significantly decreased in both treated groups as compared to controls. Aortic atherosclerotic lesion size was significantly lower in the phytosterol‐treated and significantly higher in the probucol‐treated mice as compared to controls. Plasma and aortic PAI‐1 levels were significantly higher in the probucol‐treated animals as compared to controls, while these values were comparable between the phytosterol‐treated and control mice. Both DNA microarray and RT‐PCR tests showed a significant increase in the expression of F13a1 in the liver of probucol treated mice as compared to controls. These observations suggest that pro‐atherogenic effects of probucol maybe mediated through alterations in the coagulation system in this animal model.